Proteomic Characterization of Breast Cancer Exosomes

Exosomes are small membrane-lined vesicles released by certain cells, including cancer cells and reticulocytes, into the extracellular space. Once there, they can move from cell to cell and from tissue to tissue, potentially acting as extracellular signaling moieties. Exosomes have been found in synovial fluid, breast milk, semen and urine, among other extracellular fluids.

Once released, exosomes carry proteins and RNA from cell to cell. Researchers are now investigating whether exosomes are involved in extracellular signaling between malignant and non-malignant cells. There is speculation that exosomes can enter the circulation and affect non-malignant cells, thus influencing tumor progression. Exosomes are also of interest as carriers of cancer biomarkers around the body. Their existence in extracellular fluids makes them ideal candidates for biomarker discovery and non-invasive testing.

In their study, Kruger et al. (2014) used molecular and proteomics techniques to characterize exosome contents released by two breast cancer cell lines.1 By comparing results from the two cell lines, they were also able to correlate findings with the degree of malignancy exhibited by each cell line.

The researchers cultured two breast cancer cell lines, MCF7 and MDA-MB231, representative of cancers with metastatic potential. They collected exosomes secreted by the cultured cells into serum-free media, then used gradient centrifugation to collect the extracellular vesicles.

First, the researchers characterized the exosomes collected, finding them to be round vesicles of between 80 and 200 nm in diameter under electron microscopy. Once lysed, the exosome contents were separated using polyacrylamide gel electrophoresis. Following in-gel digestion, the researchers examined the tryptic digests via liquid chromatography–tandem mass spectrometry (LC-MS/MS) using an Orbitrap XL mass spectrometer (Thermo Scientific). Kruger and co-workers then used Mascot to analyze the data acquired, identifying the proteins through comparison with the SwissProt database and the NCBI human reference sequence database.

Analysis of the results revealed 59 proteins for MCF7 exosome proteomes and 88 for MDA cultures, with 27 proteins common to both cell lines. Once identified, the researchers found clear differential expression between the cell lines; MCF7 exosomes contained proteins associated with nucleic acid and protein binding roles, whereas those found in MDA exosomes were predominantly more catalytic. Matrix metalloproteinases, implicated in cancer progression, were more abundant in the MDA exosomes.

Further analysis showed a difference in the expression of extracellular matrix (ECM) proteins; 24% of proteins expressed in MDA exosomes were associated with ECM but only 8.6% of MCF7. The authors consider that these findings might reflect the higher metastatic potential of MDA cells.

Researchers confirmed the proteomics findings with Western blotting to identify and quantify exosome proteins from each cell line. In addition, they also checked results using real-time PCR amplification, showing that mRNA expression matched the proteins identified.

Kruger and co-workers also isolated exosomal micro RNA (miRNA). When they analyzed gene ontology, the researchers found several miRNAs associated with tumorigenesis, including mir-34a and mir-106b, which regulates TGFβ induction during tumor metastasis.

The authors consider that characterizing exosome contents from breast cancer cell lines and understanding their role in ongoing oncogenesis is important in cancer research. Determination of their oncogenic potential is an important aspect of biomarker discovery and for development of future therapeutic protocols.